PROJECT SUMMARY Protein homeostasis, or proteostasis, is achieved through a regulated network of the translational machinery, molecular chaperones, and protein degradation factors, which function to maintain the abundance of the cell?s proteins in folded, soluble, non-aggregated states. Deterioration of the proteostasis network is a hallmark of aging cells and underlies numerous diseases associated with aging. A key mechanism regulating factors within the proteostasis network is their post- translational modification, including methylation at lysine residues. The SMYD family of lysine methyltransferases has been shown to interact with molecular chaperones in diverse systems, and there are numerous reported lysine methylation sites on factors that contribute to proteostasis. However, our knowledge of the methyl-lysine substrates targeted by SMYD methyltransferases and their functional interactions with the proteostasis network is still very limited. The primary objective of the proposed work is to leverage proteomic and molecular tools in the model system Saccharomyces cereviasie to define the substrates for the yeast SMYD enzyme Set6, a largely uncharacterized protein, and determine its molecular function in regulating the proteostasis network. In Aim I, we will use high-resolution mass spectrometry to identify targets of Set6 proteome-wide and perform molecular and biochemical characterization of lysine methylation on the newly-identified substrates. In Aim II, we will use molecular and genetic tools to define the role of Set6 in interacting with and regulating the proteostasis network to prevent the accumulation of unfolded proteins. In total, this work will integrate proteomic and genetic analyses to advance our understanding of the mechanistic links between the SMYD family lysine methyltransferases and molecular chaperones. We expect this work to provide new insights into regulatory mechanisms critical to proteostasis, which may shed light on new targets for therapeutic interventions into diseases associated with protein misfolding and aggregation. Furthermore, these studies will also develop a well-characterized, genetic model system for dissecting the molecular mechanisms by which SMYD enzymes contribute to proteostasis and determining how their manipulation may prevent aging-associated pathologies.